Abstract
BackgroundUrokinase-type plasminogen activator (uPA) plays a major role in extracellular proteolytic events associated with tumor cell growth, migration and angiogenesis. Consequently, uPA is an attractive target for the development of small molecule active site inhibitors. Most of the recent drug development programs aimed at nonpeptidic inhibitors targeted at uPA have focused on arginino mimetics containing amidine or guanidine functional groups attached to aromatic or heterocyclic scaffolds. There is a general problem of limited bioavailability of these charged inhibitors. In the present study, uPA inhibitors were designed on an isocoumarin scaffold containing uncharged substituents.Results4-Chloro-3-alkoxyisocoumarins were synthesized in which the 3-alkoxy group contained a terminal bromine; these were compared with similar inhibitors that contained a charged terminal functional group. Additional variations included functional groups attached to the seven position of the isocoumarin scaffold. N- [3-(3-Bromopropoxy)-4-chloro-1-oxo-1H-isochromen-7-yl]benzamide was identified as an uncharged lead inhibitor of uPA, Ki = 0.034 μM. Molecular modeling of human uPA with these uncharged inhibitors suggests that the bromine occupies the same position as positively charged arginino mimetic groups.ConclusionThis study demonstrates that potent uncharged inhibitors of uPA can be developed based upon the isocoumarin scaffold. A tethered bromine in the three position and an aromatic group in the seven position are important contributors to binding. Although the aim was to develop compounds that act as mechanism-based inactivators, these inhibitors are competitive reversible inhibitors.
Highlights
Urokinase-type plasminogen activator plays a major role in extracellular proteolytic events associated with tumor cell growth, migration and angiogenesis
We have focused on the synthesis and testing of uncharged compounds as leads for the development of Urokinase-type plasminogen activator (uPA) inhibitors with improved bioavailability. 4Chloroisocoumarin was selected as the scaffold, in which substituted 3-alkoxy groups were introduced that contained neutral terminal functional groups or charged terminal functional groups [17]
In this study we demonstrate that introduction of bromine in place of a terminal charged functional group in the 3-alkoxy substituent provides uncharged uPA inhibitors with low micromolar dissociation constants
Summary
Urokinase-type plasminogen activator (uPA) plays a major role in extracellular proteolytic events associated with tumor cell growth, migration and angiogenesis. UPA is an attractive target for the development of small molecule active site inhibitors. Multiple proteases, including matrix metalloproteases (MMP-2, MMP-9 and MMP-14), cysteine proteases (cathepsin B and cathepsin L), aspartyl protease (cathepsin D) and serine proteases (plasmin, matriptase and urokinase) participate in cancer cell growth, metastasis and angiogenesis [1,2,3,4]. Many cancer cells secrete pro-uPA and its receptor uPAR; binding of pro-uPA to uPAR leads to its activation, with subsequent generation of plasmin by the uPA-catalyzed hydrolysis of extracellular plasminogen [7,8]. UPA has become a major target for development of non-peptidic small molecule inhibitors as potential anti-cancer drugs [10,11] The surface location of bound uPA provides directionality to the degradation of matrix, thereby assisting the directional migration of cancer cells. uPA in complex with uPAR affects other biological processes including signaling pathways that influence cell proliferation [9]. uPA has become a major target for development of non-peptidic small molecule inhibitors as potential anti-cancer drugs [10,11]
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